As an essential element to all living cells, copper can be highly toxic when allowed to accumulate in excess of cellular needs. The intracellular free copper concentration is controlled below the toxic level by both copper efflux ATPases whose gene expressions are regulated by metal-sensitive transcription factors, and metallochaperones that guide copper into the target proteins and protect this highly active element from adventious actions. However, little is known about the molecular and mechanistic principles of these processes. Simple organisms such as Escherichia coli provides an excellent model system to study copper homeostasis in cells. Recent studies have shown that in E. coli a copper-responsive transcription regulator, namely CueR, is responsible for gene expression of the principal copper-exporter, CopA. This proposal focuses on the mechanistic details of the CueR-mediated transcription regulation of the CopA gene and the molecular and structural bases of CueR in metal recognition. By calibrating the copper sensitivity of the CueR/CopA gene interaction in vitro, this proposal provides a convenient probe to determine the intracellular free copper concentration in E. coli. These results may provide experimental evidence for the importance of metallochaperones in prokaryotic cells, and may lead to the discovery of the first copper chaperone in E. coli. These studies lay the groundwork for delineating the fundamental principles of metal homeostasis in both prokaryotic and eukaryotic cells.